Sequestration within peptide coacervates improves the fluorescence intensity, kinetics, and limits of detection of dye-based DNA biosensors

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality.

After 20 minutes both samples were split into two samples and one was centrifuged at 10 min at 15k rpm and the fluorescence of the supernatant (Centrifuged) was compared to the non-centrifuged (Control) sample.As can be seen the buffer sample actually had slightly higher fluorescence after centrifugation, while the remaining fluorescence in the coacervate centrifugation samples was less than 0.3 ± 0.1%.The experiment qualitatively demonstrates that the coacervates are dynamic and that the DNA and peptides are capable of moving within the structures.Quantitative estimates are determined by the Mobile Fraction as determined by the fitting process detailed below.We observe that the HP form appears to be slightly more mobile than the dsDNA, which is reasonable considering it has a lower molecular weight and is more compact.
The recovery of fluorescence was normalized using the double-normalization equation - ] Eq.S1 Where npre = number of frames pre-bleaching, I(t) Eq.S2

Figure S1 :
Figure S1: A) Absorbance spectra of the MB DNA, and a 2:1 target:MB samples (dsDNA) in buffer at 20 and 65 °C.B) Normalized absorbance of the 260 nm and or 530 nm absorbance of the MB and dsDNA samples.The 260 nm peak increases as the DNA goes from ds to ss, while the 530 nm peak decreases as the vibronic coupling of the Cy3-Cy5 due to their close-proximity diminishes upon heating.Curves are Boltzmann fits, and melting temperatures are determined at by the fitting inflection point.

Figure S2 :
Figure S2: Normalized absorbance spectra of Cy3, Cy5, and FRET pair in buffer conditions, as well as the FRET pair in Coacervate conditions.It can be observed that neither FRET pair is the linear combination of the individual dyes.This suggests that the dyes are strongly coupled, which has often been shown to lead to non-radiative decay pathways for both the donor (Cy3) and acceptor (Cy5) dye.

Figure S3 :
Figure S3: Representative data from fluorescence microscopy (Coacervates with 1:2 MB:target addition) showing the total fluorescence within the coacervates and the background.Data collected from 2 different images and 10 region of interests (ROIs).Average values are 0.06 ± 0.08 counts for background and 20.2 ± 3.3 counts for coacervates, this results in a value of 99.7 ± 0.4 % of fluorescence within coacervates.

Figure S4 :Figure S5 :
Figure S4: Fluorescence spectra obtained from confocal fluorescence microscopy of coacervates with pre-formed MB to target ratios.Images on the right are representative of the data on the left.Scale bar is 5 μm.Uncertainties arise from averaging the spectra of over 10 different images and ROI for each preparation.

Figure S6 :
Figure S6: FRAP experiments as detailed in the Methods section in main text.A) Microscopy image of coacervates with MB in HP form.B) Representative FRAP curve from sample in (A).C) Microscopy image of coacervates with MB+target in dsDNA form.D) Representative FRAP curve from sample in (C).The experiment qualitatively demonstrates that the coacervates are dynamic and that the DNA and peptides are capable of moving within the structures.Quantitative estimates are determined by the Mobile Fraction as determined by the fitting process detailed below.We observe that the HP form appears to be slightly more mobile than the dsDNA, which is reasonable considering it has a lower molecular weight and is more compact.
ROI1' and I(t) ROI2' are the fluorescence intensities of the bleached spot and the unbleached spot within the condensate respectively upon background subtraction.Normalized data is plotted as a function of time and fitted using exponential function of the form per EasyFRAP analysis manual to extract mobile fraction (mf) Ifit = I0 -a.e(-.t)-g e(-.t) Mf = + 1−(0−−) Figure S7: Control demonstrating random DNA sequence (ATATAATCGCTCGCATATTATGACTG) does not activate MB fluorescence signal change.Target and Random DNA added at 500 nM.

Figure S8 :
Figure S8: Representative kinetics of MB reacting to target strand addition in (A) buffer or (B) coacervates.The data set shown here is the average of two runs and was used, along with other similar experiments, to create the LOD Figure 6 in the main text.